Immobilized DNA polymerase

Abstract

The present invention relates to a DNA polymerase immobilized by covalent bonding. More particularly, the present invention relates to an immobilized DNA polymerase whose activity is maximally preserved by masking the active site of the DNA polymerase and optimizing interaction of the masked molecule to the substrate material. In one embodiment, the average activity of the immobilized DNA polymerase is more than about 10% relative to that of the solution phase DNA polymerase. Further provided by the invention are methods and kits for performing polymerase chain reactions (PCR).

1. An immobilized DNA polymerase comprising a DNA polymerase having an active site and an immobilization site, the immobilized DNA polymerase further comprising a linker, a matrix molecule, and a substrate material, wherein
 the DNA polymerase is bonded by a covalent bond with to the linker; 
 the linker is bonded by a chemical bond to the substrate material; 
 the immobilization site of the DNA polymerase is distant from the active site; 
 the matrix molecule comprises a non-reactive terminal group and is connected to the substrate material together with the linker, 
 and wherein the average number of covalent bonds formed between the immobilized DNA polymerase and the linker is such that average activity of the immobilized DNA polymerase is more than about 10% of the activity of solution phase DNA polymerase. 
 
     
     
       2. An immobilized DNA polymerase comprising a DNA polymerase having an active site and an immobilization site, the immobilized DNA polymerase further comprising a linker, a matrix molecule, and a substrate material, wherein
 the active site is masked with a DNA polymerase substrate; 
 the DNA polymerase is bonded by a covalent bond with to the linker; 
 the linker is bonded by a chemical bond to the substrate material; 
 the immobilization site of the DNA polymerase is distant from the active site; 
 the matrix molecule comprises a non-reactive terminal group and is connected to the substrate material together with the linker, 
 and wherein the average number of covalent bonds formed between the immobilized DNA polymerase and the linker is less than about five bonds, such that average activity of the immobilized DNA polymerase after demasking is more than about 10% of the activity of solution phase DNA polymerase. 
 
     
     
       3. An immobilized DNA polymerase as in  claim 1  or  2 , wherein the DNA polymerase is a thermostable DNA polymerase or is a DNA polymerase that is not thermostable. 
     
     
       4. An immobilized DNA polymerase as in  claim 1  or  2 , wherein the linker comprises a reaction group at one end that is capable of binding to the DNA polymerase and a reaction group at another end that is capable of binding to the substrate material. 
     
     
       5. An immobilized DNA polymerase as in  claim 1  or  2 , wherein the substrate material is selected from the group consisting of metal, nonmetal, metalloid, compounds thereof, and mixtures thereof, and has reaction groups capable of forming chemical bonds with the linker on its surface. 
     
     
       6. An immobilized DNA polymerase as in  claim 1  or  2 , wherein the covalent bond between the DNA polymerase and the linker is an amide bond between a primary amine and a carboxyl group. 
     
     
       7. The immobilized DNA polymerase of  claim 1  or  2 , wherein the mole fraction of the linker relative to the total moles of the linker and the matrix molecule is between from about 0.5% to about 50%. 
     
     
       8. The immobilized DNA polymerase of  claim 7 , wherein the mole fraction of the linker relative to the total moles of the linker and the matrix molecule is between 0.5% to about 10%. 
     
     
       9. The immobilized DNA polymerase of  claim 1  or  2 , wherein the substrate material is a filter, polymer, co-polymer, polymer blend, graft co-polymer or polymer adduct. 
     
     
       10. The immobilized DNA polymerase of  claim 9 , wherein the substrate material is selected from the group consisting of poly(ethylene glycol), poly(vinyl pyrrolidone), poly(vinyl alcohol), poly(amino acids), divinylether maleic anhydride, ethylene-maleic anhydride, N-(2-hydroxypropyl)methacrylamide, dextran and a blend thereof. 
     
     
       11. The immobilized DNA polymerase of  claim 2 , wherein the average number of the covalent bonds formed between the immobilized DNA polymerase and the linker is less than about three. 
     
     
       12. The immobilized DNA polymerase of  claim 2 , wherein the average number of the covalent bonds formed between the immobilized DNA polymerase and the linker is about one. 
     
     
       13. The immobilized DNA polymerase of  claim 1  or  2 , wherein the number of the covalent bonds formed between the immobilized DNA polymerase and the linker is controlled by adjusting the number density of the linker on the substrate to between about 2×10 12  cm −2  to 2×10 14  cm −2 . 
     
     
       14. The immobilized DNA polymerase of  claim 3 , wherein the thermostable DNA polymerase is a Taq DNA polymerase or catalytically active fragments or derivatives thereof. 
     
     
       15. The immobilized DNA polymerase of  claim 3 , wherein the DNA polymerase that is not thermostable is an  E. coli  DNA polymerase or a T7 DNA polymerase or catalytically active fragments or derivatives thereof. 
     
     
       16. The immobilized DNA polymerase of  claim 1  or  2 , wherein the DNA polymerase comprises two domains and one domain does not have polymerase activity, wherein the immobilization site of the DNA polymerase is present in the domain that does not have polymerase activity.